Atrial fibrillation (AF) is the most common cardiac arrhythmia, a condition that predisposes individuals to heart failure and stroke and is a major contributor to cardiovascular mortality. There is an unmet need to treat AF and the underlying electrical and structural changes in heart. There is a long unsuccessful history of ion channel blockers in AF; invariably they are proarrhythmic. We have identified a new target for atrial fibrillation, Ca2+/CaM-dependent protein kinase II (CaMKII), whose hyperactivity is pro-arrhythmic via multiple pathways and systems implicated in the genesis of AF and ventricular arrhythmia (VA). Targeting CaMKII is an innovative new paradigm - treating AF while also being anti-arrhythmic in ventricle. In humans and mice CaMKII made hyperactivity by autophosphorylation and oxidation elicits a diastolic Ca2+ 'leak' from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) hyperphosphorylation, leading to AF. Atrial tissue from patients with AF is marked by elevated CaMKII activity, while CaMKII inhibition prevents aberrant RyR2 Ca2+ release. Furthermore, inhibition of the kinase with our lead compound or genetic ablation of CaMKII or its phosphorylation site on RyR2 blocks this chain of events and reduces the frequency of AF in mice. We aim to modify our potent and selective lead compound to i) reduce its rapid liver microsomal metabolism in rodents, which will facilitate preclinical development toward IND, and ii) limit any central nervous system (CNS) penetration to mitigate concerns regarding CaMKII inhibition in brain. Following lead optimization we will test efficacy in an established in vivo mouse model of induced AF and in an isolated rabbit heart model. The Ryr2R176Q/+ AF mouse model exhibits a Ca2+ leak and susceptibility to ectopic activity, reentry, and AF triggered by atrial pacing. Importantly, these models share mechanisms with post-op AF, a significant inpatient indication and an achievable entry point for Allosteros Therapeutics in cardiovascular therapeutics. The study will also include assay of biomarkers to assess in vivo inhibition of CaMKII and reduction in site-specific phosphorylation of its targets, RyR2 and phospholamban. The Phase I work will position us for lead selection and IND-enabling studies and enable us to reach metabolic and drug disposition milestones sought by investors and pharma who now recognize that CaMKII is a consensus target in AF and VA.